The invention relates to a procedure and a device for fluorescence correlation spectroscopy, in particular for multicolor fluorescence correlation spectroscopy, during which light beams are focused in a transparent medium.
In multicolor fluorescence correlation spectroscopy (FCS), as described by P. Schwille et al., Biophysical Journal, Vol. 72 (1997), 1878 to 1886 or M. Vxc3x6lcker et al. tmxe2x80x94Technisches Messen, 36 (1196, 128 to 135), molecular interactions are studied by marking two reactants with different fluorescent dyes and allowing them to freely diffuse in a liquid, transparent medium. The reactants generate fluctuations in fluorescent intensity while diffusing through the focal point that can be detected with a confocal lens system. If predominantly correlated intensity fluctuates are detected between the emission wavelengths of the two fluorogens, this points to a complexing between the two partners.
Given the varying excitation wavelengths of the fluorogens, use must be made of two laser wavelengths, which are focused on the smallest possible, identical volume in the sample. As a rule, microscope lenses with a high numerical aperture, i.e., large aperture ratio, are used to this end to achieve as small a focal point as possible on the one hand, and gather as much of the emitted light
Another disadvantage to the available lens is that an optimal, diffraction-limited focal point is only ensured if the refractive index of the immersion medium and sample solution reflects the value for which the lens was optimized. Since fluorescence correlation spectroscopy is very sensitive to changes in focus volume, a change in the refractive index can have a highly adverse effect on the results. Among other things, changes in the refractive index can be caused by a change in the temperature of the sample or salts dissolved in the buffer, and hence in the parameters that are commonly varied in biochemical experiments. This problem is already encountered in single-color fluorescence correlation spectroscopy.
In terms of the procedure, the object of the invention is to develop a procedure and device that do not exhibit the described disadvantages.
This object is achieved by means of a generic procedure in which the light beams are incident on the transparent medium in an approximately perpendicular manner, and are only deflected toward the focal point inside the transparent medium.
The invention is based on the knowledge that the known errors stem from the use of refractive lens systems. The angle of incidence and angle of reflection on a surface between two media are linked by the refractive index according to Snell""s Law. If the refractive indices change owing to dispersion, i.e., a change in the refractive index with wavelength, or resulting from the use of other buffers, a change takes place in the beam path, and hence in the focus volume. This set of problems affects every beam path except for the light beams perpendicularly incident on the boundary surface. Only a light beam perpendicularly incident on the boundary surface passes the boundary surface without being deflected, independently of the refractive index. As a result of this knowledge, only reflective lens systems came to be used in the focusing arrangement to solve the task, and all light beams penetrate the boundary surfaces between different optical media only perpendicularly.
In this regard, the term xe2x80x9capproximately perpendicularxe2x80x9d is defined by the desired measuring accuracy. Deviations are possible within the framework of a desired measuring accuracy.
Hence, the described procedure enables a simple optimization of the used lens, and permits the focusing of laser beams with different wavelengths on an identical, as diffraction-limited as possible volume inside the sample.
One relatively easy way to realize the invention is to reflect the light beams inside the transparent medium toward the focal point.
In terms of the device, the object is achieved with a general device in which the sample vessel exhibits a focusing, metal-coated floor, wherein the focal point lies inside the sample vessel. In this case, the floor can be designed in such a way as to take the light beams penetrating into the sample vessel essentially parallel to each other and focus them essentially on the focal point.
This device makes it possible to use the sample vessel as a focusing element. The parallel light beams incident in the sample vessel are focused by its floor on a point. Since this point lies inside the sample vessel, there is no further deflection of the parallel incident light beams on the boundary surfaces between two media. The light beams only need to get into the sample once, and since parallel and perpendicular incident light beams are used here, they are not deflected when penetrating through the cover slip or making the transition between the cover slip and sample liquid. No boundary surfaces between different media need be overcome in the process of deflection inside the transparent sample.
It is advantageous if the sample vessel is saucer-shaped, and if the focal point lies inside the saucer. An optimal shape for the floor is achieved by giving the floor a parabolic or slightly elliptical shape.
To achieve a good measurement result, it is proposed that the floor be precisely fabricated to a fraction of the used wavelength. The high level of precision achievable with the device also requires that the metal-coated floor surface be accurately fabricated.
To ensure the long-term durability of the reflective metal coating, it is additionally proposed that the floor be metal-coated with a layer resistant to conventional buffer solutions.
Any material in which a fluorescence correlation spectroscopy can be performed may be used as the transparent medium. In particular, this can be a transparent fluid or transparent liquid or gel.